Project Summary ? Aleksander A. Rebane Imbalances in Ca2+-triggered exocytosis of hormones and neurotransmitters cause severe diseases, including type 2 diabetes and various neurological disorders such as Parkinson?s disease, schizophrenia, and epilepsy. Yet it remains poorly understood how Ca2+ triggers exocytosis. Of particular interest is synaptic exocytosis of neurotransmitters, which occurs in response to the local influx of Ca2+, triggered by the arrival of an action potential at the axonal terminal. The core machinery responsible for membrane fusion in regulated exocytosis consists of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), complexin, and synaptotagmin. SNAREs form a broad class of molecular fusion machines that associate by forcefully zippering into a coiled-coil four-helix structure, thus drawing opposing membranes into close proximity for fusion. Additional components are required to turn fusion on and off to regulate synaptic transmission. This task is achieved by complexin and synaptotagmin, which suppress unwanted spontaneous synaptic exocytosis by suspending SNARE zippering halfway as a clamp. Controlled release occurs when calcium removes the clamp to resume SNARE zippering and induce neurotransmitter release. However, the exact constituents of the clamp and the mechanism of clamping and de-clamping are poorly understood. It has been difficult to observe the molecular events along the regulated SNARE assembly pathway using traditional ensemble-based methods. These events are inherently transient and occur solely in the presence of the membrane?s repulsive force. We address these difficulties by using optical tweezers to apply precisely known pulling forces on single SNARE complex molecules to mimic membrane repulsion and to stabilize the partially assembled intermediates, while using molecular extension measurements to determine the structures on millisecond timescale and at nanometer resolution. We analyze these measurements with state-of-the-art methods to derive the conformations, energies, and kinetics of SNARE complex assembly intermediates. In Aim 1, we will analyze the effect of two mutations in the SNARE complex, SNAP-25 I67T and I67N, which cause severe neuropathy, including ataxia and intellectual disability. We will use optical tweezers to measure how these mutations change the energetics, kinetics, and pathways of SNARE zippering. We will then employ reconstituted membrane-fusion assays to compare how the observed changes in SNARE zippering affect SNARE-mediated membrane fusion. In Aim 2, we will directly observe the effect of complexin, synaptotagmin, and calcium on SNARE assembly. We will investigate by what mechanism SNARE assembly is clamped by complexin, and how synaptotagmin and Ca2+ release this clamp. Our research will reveal how SNARE mutations may cause neuropathy and how complexin and synaptotagmin regulate Ca2+-dependent release. Our research will provide concrete molecular mechanisms to act as new drug targets for neurological disease.